Dipeptidyl Peptidase I Activates Neutrophil-Derived Serine Proteases and Regulates the Development of Acute Experimental Arthritis

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Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis

April M. Adkison, … , Diane G. Kelley, Christine T.N. Pham

J Clin Invest. 2002;109(3):363-371. https://doi.org/10.1172/JCI13462.

Article

Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI–/– mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI–/– mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE–/– × CG–/– mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex–mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-α and IL-1β. These results implicate DPPI and polymorphonuclear neutrophil–derived serine proteases in the regulation of cytokine production at sites of inflammation.

Find the latest version: https://jci.me/13462/pdf Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis

April M. Adkison,1 Sofia Z. Raptis,1 Diane G. Kelley,2 and Christine T.N. Pham1, 3

1Department of Internal Medicine, 2Department of Pediatrics, and 3Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA Address correspondence to: Christine T.N. Pham, 660 South Euclid Avenue, Campus Box 8045, St. Louis, Missouri 63110-1093. Phone: (314) 362-9043; Fax: (314) 454-1091; E-mail: [email protected].

Received for publication June 7, 2001, and accepted in revised form December 19, 2001.

Leukocyte recruitment in inflammation is critical for host defense, but excessive accumulation of inflammatory cells can lead to tissue damage. Neutrophil-derived serine proteases (cathepsin G [CG], neutrophil elastase [NE], and proteinase 3 [PR3]) are expressed specifically in mature neutrophils and are thought to play an important role in inflammation. To investigate the role of these proteases in inflammation, we generated a mouse deficient in dipeptidyl peptidase I (DPPI) and established that DPPI is required for the full activation of CG, NE, and PR3. Although DPPI–/– mice have normal in vitro neutrophil chemotaxis and in vivo neutrophil accumulation during sterile peritonitis, they are protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen. Specifically, there is no accumulation of neutrophils in the joints of DPPI–/– mice. This protective effect correlates with the inactivation of neutrophil-derived serine proteases, since NE–/– × CG–/– mice are equally resistant to arthritis induction by anti-collagen antibodies. In addition, protease-deficient mice have decreased response to zymosan- and immune complex–mediated inflammation in the subcutaneous air pouch. This defect is accompanied by a decrease in local production of TNF-α and IL-1β. These results implicate DPPI and polymorphonuclear neutrophil–derived serine proteases in the regulation of cytokine production at sites of inflammation. J. Clin. Invest. 109:363–371 (2002). DOI:10.1172/JCI200213462.

Introduction neutrophil-derived proteases at sites of inflammation Polymorphonuclear neutrophils (PMNs) play a critical is to degrade various components of the extracellular role in host defense against bacterial pathogens. PMNs matrix. The role of serine proteases in PMN recruit- elaborate a number of proteases that are involved in ment, however, appears to be more complex than sim- host immunity and are capable of bactericidal activity ple disruption of extracellular matrix. Recent evidence (1, 2). However, proteolytic enzymes released by PMNs suggests that extracellularly released serine proteases also degrade extracellular matrix components, leading remain catalytically active (9), and may modulate neu- to tissue damage and chronic inflammation (3, 4). For trophil recruitment. Inhibition of PMN-derived serine example, PMNs are a prominent feature of joint proteases has been shown to reduce PMN adhesion to inflammation in rheumatoid arthritis, and are thought various extracellular matrix–coated surfaces and to be responsible for the cartilage destruction in this endothelial layers ex vivo (10, 11), and neutrophil infil- disease. Therefore, the prevention of PMN migration tration into inflamed tissues in vivo (12, 13). However, and accumulation at inflammatory sites may limit studies using naturally occurring protease inhibitors, their host-damaging effector functions. as well as low-molecular-weight inhibitors, have shown PMNs express a family of neutral serine proteases in that serine proteases are not required for successful their azurophil (primary) granules. These PMN-specif- PMN transendothelial migration in vitro (14), nor are ic, granule-associated enzymes include cathepsin G they involved in the disruption of subendothelial base- (CG), neutrophil elastase (NE), and proteinase 3 (PR3) ment membrane during PMN diapedesis (15). (5). Another structurally related granule-associated Recently, proteolysis has emerged as an important protein is azurocidin, which has no enzymatic activity mechanism of regulating cytokine bioactivity (16). For (6). While their protective role in host immunity is example, IL-1β and TNF-α are proteolytically processed acknowledged (7, 8), the role of PMN-derived serine to their active form by IL-1β–converting enzyme (ICE, proteases in neutrophil extravasation at sites of inflam- or caspase-1) and TNF-α–converting enzyme (TACE), mation is still controversial. respectively (17, 18). At sites of inflammation, elevated Due to their proteolytic properties, it has long been levels of extracellularly released, neutrophil-derived ser- commonly thought that the main function of these ine proteases temporally coincide with high concentra-

The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3 363 tions of bioactive cytokines. This led to the hypothesis cinyl-Ala-Ala-Pro-Val-pNA (Elastin Products Co., that PMN-derived serine proteases may control inflam- Owensville, Missouri, USA); CG activity was measured matory processes through the proteolytic modification using the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA and release of cytokines. However, the extent of their (Sigma-Aldrich, St. Louis, Missouri, USA); and PR3 regulatory functions in various pathophysiological con- activity was measured using the substrate N-t-buty- ditions remains largely uncharacterized. loxycarbonyl-Ala-Ala-Nva-thiobenzyl ester (Elastin Serine proteases share a high degree of homology, both Products Co.), as previously described (23). at the cDNA and protein levels. Genetic analysis shows In vitro chemotaxis. Neutrophils were purified from that all of these serine proteases are synthesized as pre- bone marrow using a discontinuous Percoll gradient proenzymes with a residue leader sequence of 18–26 (Amersham Pharmacia Biotech, Piscataway, New Jersey, amino acids that is cleaved, leaving a prodipeptide USA). Neutrophil purity was consistently 75–85%, as between the proenzyme and the mature active enzyme assessed by light microscopy of Wright-Giemsa–stained (19). The activation of these proteases requires the cytospins. Neutrophils were labeled with calcein AM removal of this prodipeptide (20). We recently generated (Molecular Probes Inc., Eugene, Oregon, USA), and a mouse with a null mutation in the lysosomal cysteine placed on top of a 96-well chemotaxis chamber (Neuro protease dipeptidyl peptidase I (DPPI), and showed that Probe Inc., Gaithersburg, Maryland, USA). The bottom DPPI is essential for the processing and activation of wells of the microplate were filled with either the many serine proteases, including cytotoxic lymphocyte chemotactic agent fMLP (10–4 M), or zymosan-activat- granzymes A and B (21) and mast cell chymase (22). ed rat serum (2%, as a source of C5a), or recombinant In this study, we established that DPPI is important for human IL-8 (rhIL-8) (3 µg/ml; Amersham Pharmacia the processing and activation of the PMN-derived serine Biotech), or buffer. After incubation for 1 hour at 37°C proteases NE, CG, and PR3. Although DPPI- and serine in an atmosphere of 5% CO2, the nonmigrating cells protease–deficient mice have normal in vitro chemotaxis from the top of the filter were removed, and the cells and in vivo peritonitis in response to thioglycollate, they (tested in triplicate per point) that migrated to the bot- are resistant to acute arthritis induction by passive trans- tom chamber were measured using a fluorescent plate fer of anti–type II collagen antibodies. Moreover, they reader (Bio-Tek Instruments Inc., Winooski, Vermont, have a defect in neutrophil recruitment in subcutaneous USA). The fluorescence for each well was expressed as a air pouches in response to zymosan and immune com- percentage of total PMNs. plexes. The decrease in inflammatory cell accumulation Superoxide production. Purified bone marrow–derived correlates with a decrease in cytokine production. These neutrophils were resuspended in HBSS containing studies support the hypothesis that neutrophil-derived 1.3 mM CaCl2 and 0.4 mM MgSO4, and stimulated serine proteases are involved in the regulation of with PMA in the presence of 0.2 mM cytochrome c. cytokine production at sites of inflammation. The kinetics of superoxide dismutase–inhibitable reduction of cytochrome c was assessed by measuring – 6 Methods nmol of O2 produced by 10 PMNs over a period of –/– –/– –/– –/– –/– Animals. DPPI , CG , NE , and NE × CG mice, 30 minutes, at OD550. all in the 129 genetic background, were produced as Thioglycollate-induced peritonitis. Mice were injected previously described (7, 21, 23). These mice and wild- with 1 ml of thioglycollate broth intraperitoneally, and type (WT) control mice were kept in pathogen-free con- at the indicated timepoints, peritoneal cells were har- ditions at Washington University Specialized Research vested by lavage and quantified. Facility. For in vitro analysis, mice 4–8 weeks of age Arthritis induced by anti–type II collagen antibody. Mice were were used. For in vivo experiments, male mice 6–8 injected intravenously with a cocktail of 4 mg of weeks of age were used. anti–type II collagen antibodies (Chemicon Internation- Western blot analysis. Protein lysates were prepared al Inc., Temecula, California, USA) on day 0, followed by from PMNs or bone marrow as previously described 50 µg of intraperitoneal LPS on day 3 as recommended (23). Equivalent amounts of total protein were loaded by the manufacturer. Severity of arthritis in each paw was onto 10% SDS-PAGE gels, transferred to nitrocellulose, scored as follows: 0 = normal; 1 = mild redness and and blotted with anti-NE and anti-CG antisera as pre- swelling of digits, ankle, or wrist; 2 = moderate swelling viously described (7, 23). β-actin served as control for of ankle or wrist; 3 = severe swelling involving entire paw, quantity and quality of protein. including digits (maximum score of 12 per mouse). S1 analysis. Total cellular RNA was prepared from Histological grading of arthritis. At least four mice per bone marrow cells and analyzed by S1 nuclease protec- genotype were sacrificed, and all four paws of each tion assay using CG-specific probe as previously mouse were excised and fixed in 10% buffered formalin. described (23). Murine β2 microglobulin served as con- Fixed tissues were decalcified in 14% EDTA for 2 weeks, trol for quantity and quality of RNA. and then dehydrated and embedded in paraffin. Sagittal Determination of enzymatic activity. Total bone marrow sections of talocrural or radiocarpal joints were stained cell lysates were prepared as previously described (23) with either hematoxylin and eosin or toluidine blue and normalized for protein content. NE activity was using standard techniques. Cells infiltrating the synovial measured using the peptide substrate N-methoxysuc- and subsynovial space were identified by morphology

364 The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3 and photographed using the LE-Digital 1-CCD video In vitro neutrophil degranulation. In vitro degranulation camera system (Optronics, Goleta, California, USA). Cell assays were performed as previously described (25). counts and differentials of four random high-power Bone marrow–derived neutrophils were resuspended in fields (HPFs) of each joint section were performed by the HBSS at 107/ml, and stimulated with 50 ng/ml PMA same blinded observer. Joint exudates were scored on a for 15 minutes. The cells were pelleted, and the super- scale of 0 (normal) to 5 (severe, with presence of inflam- natant was analyzed by gelatin zymography for matrix matory cells fibrin-like material occupying more than metalloproteinase 9 (MMP-9) activity. Briefly, the 60% of the joint space). Proteoglycan depletion was supernatant was subjected to SDS-PAGE on gels con- scored on toluidine blue–stained sections on a scale of taining 1% gelatin under nonreducing conditions. 0–3 (0 = fully stained cartilage, 1 = less than 25% After electrophoresis, gels were washed twice with 2.5% unstained cartilage, 2 = 25–50% unstained cartilage, and Triton X-100 for 30 minutes to remove SDS, and then 3 = more than 50% unstained cartilage). incubated overnight at 37°C in reaction buffer con- Subcutaneous air pouch. The air pouches were formed taining 50 mM Tris (pH 7.4), 150 mM NaCl, and 5 mM according to the method of Edwards et al. (24). Mice CaCl2. Gelatinolytic activity appeared as colorless were anesthetized, and air pouches were created by sub- bands against a blue background when stained with cutaneous injection of 5 ml of sterile air into the back. 0.125% Coomassie brilliant blue. The pouches were reinflated 3 days later with 3 ml of Statistical analysis. Wilcoxon’s two-tailed rank sum sterile air. On day 6, the pouches were instilled with test for unpaired variables was used to compare dif- either 1 µg of rIL-8, 0.3 ml of 1% zymosan, or normal ferences between groups. P values of less than 0.05 saline. At the indicated times, the mice were sacrificed, were considered significant. the pouches were lavaged with 5 ml of PBS, and the number of PMNs was determined. Results Reverse passive Arthus reaction. Mice were injected intra- DPPI–/– mice have normal granulocyte development. We venously with 20 mg/kg chicken ovalbumin (OVA), fol- analyzed the hematopoietic development of multiple lowed by instillation of 300 µg heat-inactivated rabbit DPPI–/– mice derived from two different embryonic anti-OVA antibodies (Sigma-Aldrich) in the air pouch stem cell lines. Complete blood counts and differen- to induce immune complex formation in situ. Mice tials showed normal counts of circulating white blood treated with normal saline in the air pouch after intra- cells (WBCs) and mature granulocytes (PMNs) in venous injection of OVA served as controls. At indicat- both WT (9.08 ± 2.7 × 103 WBCs/µl with 24.7% ± 7.7% ed times after challenge, mice were sacrificed, and their PMNs) and DPPI–/–animals (8.79 ± 3.48 × 103 pouches were lavaged as above. WBCs/µl with 24.4% ± 10.9% PMNs). Cytospins of Induction and measurement of cytokines. Mice were bone marrow cells derived from 4- to 8-week-old WT injected intraperitoneally with 50 µg or 100 µg of E. coli and DPPI–/– mice revealed normal numbers of myeloid LPS, serotype 0127:B8 (Sigma-Aldrich), in 100 µl of precursors and mature granulocytes (data not sterile PBS. Sera were collected before and 90 minutes shown). Transmission electron microscopy of bone after LPS challenge. TNF-α levels were determined marrow–derived PMNs from DPPI–/– mice showed using an ELISA kit (R&D Systems Inc., Minneapolis, normal numbers of electron-dense azurophil granules Minnesota, USA) per the manufacturer’s protocol. and normal neutrophil morphology (data not TNF-α and IL-1β levels in air pouch supernatants col- shown). In addition, we have previously shown that lected at 12 hours after injection were also measured by PMNs derived from DPPI–/– mice express normal sur- ELISA as recommended by the manufacturer. face levels of Gr-1 and Mac-1 (21).

Figure 1 Analysis of PMN-derived serine proteases in DPPI–/– mice. NE (a) and CG (b) protein expression in DPPI–/– bone marrow lysates. β-actin served as control for protein content. Note the markedly reduced level of immunoreactive CG in DPPI–/– bone marrow lysate compared with WT. (c) S1 nuclease protection assay revealed equivalent levels of CG mRNA in DPPI–/– and WT bone marrow lysate. β2 microglobulin (β2m) served as control for RNA load- ing. (d) DPPI–/– bone marrow cells and NE–/– bone marrow cells have equivalent residual levels of NE activity. (e) Both DPPI–/– and CG–/– bone marrow cells have no detectable hydrolysis of the CG-specific peptide substrate. (f) Conversion of the PR3 substrate by DPPI–/– bone marrow cell lysate was reduced by 80% compared with WT. Relative enzyme activity was measured at OD405 per 105 bone marrow cells and values represent mean activity ± SEM of at least three animals.

The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3 365 tually absent in DPPI–/– bone marrow lysate (Figure 1e). Lastly, PR3 activity, as assayed by the hydrolysis of N-t- butyloxycarbonyl-Ala-Ala-Nva-thiobenzyl ester, was reduced by 80% in DPPI–/– lysate (Figure 1f). It is diffi- cult to assess whether this residual amount of activity is due to low levels of active PR3 enzyme, since PR3-deficient mice are currently unavailable. Since chromogenic substrates are not entirely specific for a given enzyme, it is likely that the apparent detection of residual NE and PR3 activities in DPPI–/– bone marrow is attributable to irrelevant proteases. However, we have previously shown that granzyme C and mast cell tryptase can be processed to the active form in the absence of DPPI (21, 22). There- fore, sensitive immunological assays that specifically detect the mature form of each of the three serine proteases will be necessary to firmly establish that DPPI–/– neutrophils lack all mature CG, NE, and PR3. Never- theless, these results confirmed that DPPI plays an important role in the processing and full activation of Figure 2 all three PMN-derived serine proteases in vivo. In vitro PMN function and in vivo response to thioglycollate in the DPPI–/– mice have normal in vitro neutrophil chemotaxis and absence of DPPI. (a) Bone marrow–derived PMNs from WT and in vivo thioglycollate-induced peritonitis. Neutrophil chemo- DPPI–/– mice were allowed to migrate across a filter in response to 10–4 M fMLP, 2% zymosan-activated rat serum (ZAS), or 3 µg/ml taxis was examined using a modified Boyden chamber. rhIL-8. The number of cells that migrated was expressed as a per- Calcein AM–labeled PMNs were allowed to migrate in centage of total PMNs. Values represent the mean ± SEM of at least response to fMLP (10–4 M), zymosan-activated rat serum three animals. (b) Superoxide production by PMNs in response to (2%), and rhIL-8 (3 µg/ml). There was no significant dif- – 6 PMA, expressed as nmol of O2 generated per 10 PMNs (n > 3 per ference in the ability of DPPI–/– PMNs to migrate toward genotype). The number of PMNs and macrophages recruited in these stimuli compared with that of WT PMNs (Figure response to intraperitoneal injection of thioglycollate is normal in 2a). In addition, DPPI–/– PMNs mobilized a normal res- –/– DPPI mice at 4 hours (c) 24 hours (d) and 120 hours (e).The piratory burst in response to PMA, as determined by the slightly lower number of macrophages at 120 hours after thioglycollate injection in the DPPI–/– mice did not reach statistical significance kinetics of superoxide dismutase–inhibitable reduction (n = 4–5 mice per genotype). Mph, macrophages. of cytochrome c (Figure 2b). The response of DPPI–/– mice to thioglycollate- induced peritonitis was also assessed. Groups of 4–5 Serine protease activity is severely reduced in DPPI–/– neu- mice were injected intraperitoneally with 1 ml of thio- trophils. Western blot analysis using NE-specific poly- glycollate, and the peritoneal exudates were collected clonal rabbit anti-mouse sera confirmed equal after 4 hours, 24 hours, and 120 hours. Thioglycollate immunoreactive NE bands in WT and DPPI–/– PMN injection into the peritoneum resulted in equivalent lysates (Figure 1a). However, the amount of immunore- numbers of total leukocytes and PMNs recovered from active CG was markedly reduced in DPPI–/– lysates (Fig- WT mice and DPPI–/– mice at 4 hours and 24 hours ure 1b), despite normal levels of mRNA detected by S1 (Figure 2, c and d). Peritoneal lavage at 120 hours after analysis (Figure 1c). To rule out the possibility that this thioglycollate injection revealed no statistically signif- decreased level was due to nonspecific proteolytic activ- icant difference between the numbers of WT and ity that occurred after the cells were lysed, we per- DPPI–/– macrophages (Figure 2e). Thus, although the formed immunohistochemistry on cytospin samples of absence of DPPI severely reduced the activity of NE, bone marrow cells using the CG-specific antibody. We CG, and PR3 in PMNs, the lack of serine protease activ- confirmed that immunoreactive levels of CG were ity did not affect in vitro neutrophil chemotaxis or in reduced in DPPI–/– PMNs prior to cell lysis (data not vivo recruitment of leukocytes in response to thiogly- shown). Therefore, DPPI not only activates these pro- collate, a nonspecific inflammatory stimulus. teases but may influence the total levels of these DPPI–/– mice are resistant to induction of arthritis by passive enzymes in some instances. transfer of anti–type II collagen antibodies. To further Using chromogenic substrates, we measured the spe- explore the role of DPPI and PMN-associated serine cific activity of each granule-associated serine protease protease in a more pathophysiological condition, we expressed in the myeloid compartment. NE activity in challenged the DPPI-deficient mice to a well-estab- DPPI–/– bone marrow lysate, as assessed by measuring lished model of acute experimental arthritis (26). Four the hydrolysis of N-methoxysuccinyl-Ala-Ala-Pro-Val- milligrams of a cocktail of four separate monoclonal pNA, was equivalent to that seen in the NE–/– bone mar- antibodies specific for the main arthrogenic determi- row extract (Figure 1d). CG activity, as assayed by the nants of type II collagen were injected intravenously hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-pNA, was vir- into groups of WT and DPPI–/– mice. On day 3, 50 µg

366 The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3 of LPS was injected intraperitoneally into each mouse. To rule out the possibility that the absence of DPPI may LPS, a strong inducer of proinflammatory cytokines, adversely affect the release of granule-associated serine has been shown to act synergistically in the induction proteases, we performed in vitro degranulation experi- of arthritis (26). Joint inflammation developed within ments. Upon stimulation with 50 ng/ml of PMA, WT 24–48 hours after LPS injection, and peaked around and DPPI–/– neutrophils released similar levels of day 6–7. By day 7, 87.5% of WT mice (n = 16) developed MMP-9, as determined by gelatin zymography (Figure clinically evident arthritis, with a mean arthritis score 4a). MMP-9 is one of the major proteases released by neu- of 6.3 ± 3.3 (maximum score, 12 per mouse). Arthritis trophils upon activation, and is unaffected by the DPPI is manifested by redness and swelling of the entire paw, mutation. These results suggest that DPPI–/– neutrophils including digits (Figure 3a). In contrast, none of the undergo normal degranulation with stimulation. DPPI–/– mice (n = 6) displayed any clinical signs of inflammation in either joints or digits (Figure 3b). We performed histological analysis to more precisely determine the nature of the infiltrating cells. The front and hind paws of WT and DPPI–/– mice were removed and processed. Hematoxylin and eosin–stained joint sections from WT mice revealed extensive infiltration of the subsynovial tissues by inflammatory cells (91 ± 17 cells/HPF). PMNs were the predominant invading cells (78%), with a smaller contribution from macrophages and lymphocytes (Figure 3, c, e, and g). In contrast, very few inflammatory cells (9.1 ± 2.2/HPF, P < 0.001) infiltrated the subsynovial space of DPPI–/– joints (Figure 3, d, f, and h), and the majority of these cells had macrophage-like morphology. Inflammation was also assessed by the severity of the inflammatory exudate in the joint space, and by proteoglycan depletion on toluidine blue–stained sections. Abundant PMNs were also found in the joint space of WT mice, along with marked fibrin-like deposition (Figure 3, c and e). The inflammatory exudate adhered to the joint surface, which led to proteoglycan depletion, as evidenced by the loss of toluidine blue staining (Figure 3i). In contrast, DPPI–/– joint sections showed no or very scant exudate (Figure 3, d and f), with preservation of proteoglycan content in cartilage (Figure 3j). These results suggest that DPPI–/– joints were protected from PMN infiltration and cartilage destruction. In this model of acute arthritis, cartilage-bound anti–type II collagen antibodies activate the classical pathway of the complement cascade, leading to the generation of activated complement components (27). Using immunohistochemistry, we confirmed that there were equivalent amounts of anti–type II collagen antibody and C3 deposition (and activation) Figure 3 on the cartilage surface of WT and DPPI–/– knee Clinical and histological assessment of monoclonal antibody–induced arthritis. Forepaw of a WT mouse (a) and a DPPI–/– mouse (b) on day joints after intravenous injection of monoclonal anti- 7 after receiving arthrogenic antibodies against type II collagen. Note bodies (data not shown). To further confirm that the the redness and swelling involving the entire paw, including the digits, activation of the late complement components of the WT mouse (a). Histological analysis of sections taken from the C5–C9 occurred normally in both genotypes, we per- radiocarpal joints of immunized animals, stained with hematoxylin and formed hemolytic assays using sensitized sheep ery- eosin (c–h), and toluidine blue (i and j) (to detect proteoglycan). WT throcytes, and sera from WT and DPPI–/– mice. We mice (c, e, g, and i) have marked leukocyte infiltration (*) in the sub- found that serum from DPPI–/– mice can activate the synovial tissue and joint space, adherence of inflammatory cells to joint complement cascade and induce red blood cell lysis surfaces, and proteoglycan loss as evidenced by the reduction in metachromasia at the superficial border of the cartilage matrix (arrow- to the same extent as WT serum (data not shown). heads). The infiltrating cells are predominantly neutrophils (N) (g). –/– Therefore, the lack of inflammation in the DPPI DPPI–/– mice were protected from arthritis, as evidenced by normal joint mice is not due to a defect in antigen (type II colla- histology, lack of inflammatory infiltrates, and preservation of proteo- gen) recognition by the antibody, or a defect in the glycan content (d, f, h, and j). Magnification: c and d, ×16; e and f, ×40; activation of the complement cascade. g and h, ×400. E, exudate; C, carpal bones; R, radius; JS, joint space.

The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3 367 inflammatory cells infiltrating the subsynovial tissue space than did WT mice (NE–/– mice, 19 ± 8.4 cells/HPF, P < 0.001; CG–/– mice, 42 ± 29 cells/HPF, P < 0.01). In addition, both NE–/– and CG–/– joints showed less exudate and fibrin-like deposition in the joint space, along with some preservation of proteoglycan content in cartilage (Figure 5, c and d). The fact that the single protease–deficient mice showed an intermediate phenotype indicates that both NE and CG contribute to the recruitment of inflammatory Figure 4 cells, and that the effect is additive. –/– In vitro neutrophil degranulation and TNF-α production in DPPI–/– DPPI mice have decreased response to zymosan- and mice. (a) Purified PMNs (107/ml) were incubated with 50 ng/ml PMA immune complex–induced inflammation in the subcutaneous for 15 minutes, and cell-free supernatants were analyzed by gelatin air pouch. To further elucidate the mechanism(s) under- zymography. Note that DPPI–/– and WT PMNs released equivalent lev- lying the joint-protecting effect observed in the protease- els of MMP-9. (b) TNF-α production 90 minutes after intraperitoneal deficient mice, we used the air pouch model of inflam- µ α injection of 100 g LPS. Open circles represent TNF- levels prior to mation. Air pouches formed by subcutaneous injection α LPS injection. Serum TNF- levels (filled circles) after LPS injection of air into the back of a mouse develop a lining layer were equivalent in WT mice and DPPI–/– mice. resembling synovial membrane (24). The air pouch model therefore provides a suitable space to investigate In addition, a recent report suggests that mice defi- the inflammatory responses in synovial-like tissues. cient in both NE and CG are more resistant than WT Injection of zymosan into air pouches induced a neu- mice are to the effects of LPS (8). Since TNF-α has been trophil influx that peaked at 12 hours (29). This identified as the central cytokine released upon LPS stimulation, and plays a critical role in inflammatory joint disease (28), the possibility exists that reduced serum TNF-α levels in DPPI–/– mice upon LPS challenge is responsible for the resistance to arthritis. How- ever, we found that 90 minutes after intraperitoneal injection of 100 µg of LPS, WT and DPPI–/– mice had released similar levels of TNF-α into the serum (Figure 4b). We also performed the experiment using 50 µg of LPS, with similar results (data not shown). Thus, the lack of inflammation in DPPI–/– mice is not due to a decrease in serum level of TNF-α in response to sys- temic administration of LPS. Resistance to acute arthritis in DPPI–/– mice correlates with the intrinsic defect in PMN-derived serine protease activity. DPPI–/– mice have defects in many immune effector cell functions that may contribute to the observed phenotype. To further evaluate the mechanisms involved in the resistance of susceptibility, we tested whether the absence of NE and/or CG also conferred resistance to arthritis. Following injection with anti–type II collagen monoclonal antibodies and LPS, all CG–/– mice developed clinical arthritis, with a relatively low mean arthritic score (3.3 ± 1.5, P < 0.1). NE deficiency alone considerably attenuated the phenotype; only 75% of Figure 5 –/– NE mice developed clinically evident arthritis, with a Summary of clinical arthritis and histological scores of immunized mice. mean score of 1.8 ± 1.7 (P < 0.02). Similar to the DPPI–/– (a) Groups of mice with different genotypes were immunized with mice, the NE–/– × CG–/– mice were all clinically resistant arthrogenic antibodies on day 0, followed by LPS on day 3. Each paw to the induction of arthritis (Figure 5a). was evaluated and scored on a scale of 0–3, as outlined in Methods. Histological analysis confirmed the scant presence Results are expressed as the mean arthritis score on day 7 of all four of inflammatory cells in the subsynovial space of paws (maximum score of 12 per mouse). Joint sections were evaluated by a blinded observer for cellular infiltrate (b), severity of joint exudate NE–/– × CG–/– mice (5.8 ± 2.2 cells/HPF, P < 0.001) (c), and proteoglycan (PG) depletion(d). Degree of proteoglycan deple- compared with WT mice (91 ± 17 cells/HPF), little if tion was scored on a scale of 0–3 with 3 being most severe. Results are any exudate in the joint space, and no proteoglycan expressed as mean ± SEM for each of the histological features, quanti- loss (Figure 5, b–d). In accordance with the clinical fied in WT, DPPI–/–, NE–/– × CG–/–, NE–/–, and CG–/– mice (n = at least data, NE–/– mice and CG–/– mice both had fewer four animals and 16 paws per genotype). *P < 0.01, **P < 0.001.

368 The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3 NE–/– × CG–/– mice, compared with those of WT mice (Table 1). Injection of rhIL-8, a chemokine specific for neutrophils, into air pouches led to comparable levels of PMNs recruited in WT and DPPI-deficient mice (3.0 ± 1.2 × 106 and 3.8 ± 2.7 × 106 PMNs/ pouch, respectively, n = 5 per genotype) (Figure 6c). Taken together, these results suggest that defective local production of cytokines (and probably chemokines) con- tributes to impaired neutrophil recruitment at sites of inflammation in DPPI–/– mice and NE–/– × CG–/– mice.

Discussion In this report, we establish that DPPI is essential for the full activation of CG, NE, and PR3 in vivo. Although cas- cades of proteolytic activation have been proposed and confirmed for several series of proteases, it is unlikely that DPPI initiates a sequence of events during which DPPI activates CG, which in turn activates NE, which then activates PR3. Using specific serine protease–deficient mice, we have previously shown that CG–/– neutrophils have a normal level of NE activity (23); similar- ly, NE–/– neutrophils have normal CG activity (25). Since NE, CG, and PR3 activities are all severely reduced in DPPI–/– neutrophils, previous reports and this study sug- Figure 6 gest that all three serine proteases are substrates of DPPI. Neutrophil recruitment in air pouch model. Neutrophil recruitment In addition, the activation of these proteases requires in response to zymosan (ZS) (a), immune complex (IC) formation cleavage after a glutamic acid residue, a specificity that (b), and rhIL-8 (c). Mice were sacrificed 12 hours after injection of ZS and IC formation and 4 hours after rhIL-8. Each point represents is different from the known specificity of each of these –/– the number of PMNs in an individual pouch. NS, normal saline. three serine proteases (30). We also found that DPPI PMNs contain significantly lower amounts of immunoreactive CG than are found in WT PMNs, but response was reduced by 70% in the DPPI–/– mice levels of steady-state mRNA that are basically equivalent [(11.1 ± 5.1) × 106 PMNs/pouch, n = 4, P < 0.01] com- to WT levels. This result is consistent with the previous pared with WT mice [(37.7 ± 10.3) × 106 PMNs/pouch, observation that unprocessed granzyme A is more sus- n = 4] (Figure 6a). Neutrophil recruitment was also defec- ceptible to degradation in the absence of DPPI (21). In tive in the NE–/– × CG–/– mice [(18.0 ± 9.0) × 106 PMNs/ addition, DPPI also influences the total amount of active pouch, n = 5, P < 0.02]. A similar defect in neutrophil tryptase in bone marrow–derived mast cells (22). Previ- recruitment was seen with the reverse passive Arthus ous studies have shown that processing of neutrophil- reaction. WT mice treated with both intravenous OVA derived serine proteases is either part of the process of, and 300 µg of anti-OVA in the air pouch to induce in situ or the result of, the sorting of these proteases to formation of immune complexes developed a moderate azurophil granules, where these enzymes are stored in but significant neutrophil accumulation in the pouches their active form (31). However, sorting may also occur [(2.0 ± 0.9) × 106 PMNs/pouch, n = 5] measured 12 hours normally in the absence of the prodipeptide (32). There- after injection (Figure 6b). The number of emigrated fore, it is likely that in the absence of DPPI, these PMNs was reduced in DPPI–/– mice [(0.9 ± 0.5) × 106 PMNs/pouch, n = 5, P < 0.1] and NE–/– × CG–/– mice [(0.6 ± 0.3) × 106 PMNs/pouch, n = 4, P < 0.05] (Figure Table 1 –/– –/– × –/– 6b). In fact, the response of NE–/– × CG–/– mice to the Defective cytokine production in DPPI and NE CG mice treatment was equivalent to their response to injection Genotype Treatment TNF-α IL-1β of normal saline into air pouches [(0.7 ± 0.1) × 106 (pg/ml) (pg/ml) PMNs/pouch, n = 4]. WT Saline 11 ± 2 9 ± 1 To investigate whether local cytokine production is Zymosan 60 ± 9 899 ± 137 normal in this model, we measured levels of TNF-α and DPPI–/– Saline 11 ± 2 33 ± 9 A B IL-1β in the air pouches following zymosan injection. Zymosan 35 ± 7 538 ± 144 –/– × –/– –/– –/– × –/– NE CG Saline 11 ± 1 12 ± 4 Although DPPI mice and NE CG mice chal- Zymosan 30 ± 1C 542 ± 199B lenged systemically with LPS showed normal levels of TNF-α production in serum (ref. 8 and Figure 4b), we Groups of 4–5 mice were injected with 0.3 ml of 1% zymosan or normal saline α β into air pouches created 6 days before. Animals were sacrificed 12 hours found that local production of TNF- and IL-1 was later, and the lavage fluid was assayed for the specified cytokine. AP < 0.01, decreased in air pouches of both DPPI–/– mice and BP < 0.02, CP < 0.001.

The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3 369 enzymes persist in a proform that can be missorted into ine proteases to regulatory functions in the cytokine net- a different granule compartment (32). Neutrophils work. Although DPPI–/– mice and NE–/– × CG–/– mice pro- express several classes of granules (azurophil, specific, duced normal serum levels of TNF-α in response to sys- gelatinase, and secretory), and missorting of azurophil temically administered LPS (ref. 8 and Figure 4b), the granule serine proteases potentially targets them for local cytokine response in the air pouch was reduced by degradation. The fact that the CG level is reduced while approximately 50% compared with the response in WT the NE level is not also suggests that individual granule mice. In addition, injection of IL-8, a chemokine specific proteases may not be sorted in an identical manner. for neutrophils, led to an equivalent number of PMNs Although recent reports suggest that membrane- recruited into DPPI-deficient air pouches. Taken togeth- bound serine proteases play a role in chemotaxis (33, 34), er, these results support the concept that DPPI- and ser- we found that DPPI–/– PMNs migrate normally in vitro in ine protease–deficient PMNs do not have an intrinsic response to optimal concentrations of various chemoat- defect in their ability to transmigrate across the endothe- tractants. In addition, using a model of nonspecific lial barrier into the air pouch environment in response to inflammation induced by intraperitoneal injection of CXC-like chemokines. Instead, the findings are in keep- thioglycollate, we showed that leukocyte recruitment into ing with the concept that serine proteases modulate the the peritoneum is normal in these mutant mice. These local release and/or processing of cytokines, and possibly observations are in agreement with recent studies that of chemokines. However, the possibility remains that report normal neutrophil chemotaxis and neutrophil DPPI–/– PMNs and NE–/– × CG–/– PMNs respond differ- accumulation during sterile and infectious peritonitis in entially to other chemoattractants that are generated CG–/–, NE–/–, and NE–/– × CG–/– mice (7, 8, 23). locally in the inflamed joint. Recent evidence suggests that PMN-derived serine Alternatively, PMNs may preferentially use proteases to proteases are also expressed on the neutrophil surface, infiltrate certain extracellular matrix–rich environments and are active and protected from naturally circulating (such as the joints) during specific inflammatory inhibitors (9). This concentrated expression of serine responses. In the extravascular space, directional cell proteases on the surface of PMNs could be important motility depends on both adhesion and subsequent for the processing of cytokines. Several studies suggest detachment. The extracellular expression of proteases that PMN-derived serine proteases are involved in the could represent a general mechanism used by various cell control of cytokine production. PR3 has been shown to types to regulate locomotion. For example, the binding be a potent activator of membrane-bound TNF-α and of urokinase-type plasminogen activator (uPA, a serine pro–IL-1β (35). CG, NE, and PR3 have been reported to protease) to its receptor is an important system regulat- catalyze the release of more active forms of CXC ing cell migration and invasiveness in normal and patho- chemokines, such as IL-8 and epithelial cell–derived logical conditions (40). The matrix metalloprotease neutrophil activating protein-78 (ENA-78), through MMP-2 colocalizes with the surface integrin αvβ3 on proteolytic processes (36, 37). In addition, PR3 and NE angiogenic blood vessels, and has been shown to play an also enhance the production of IL-8 by endothelial cells essential role in angiogenesis by promoting cell migration (38, 39). Therefore, it is reasonable to hypothesize that (41). Recent evidence suggests that PMN-derived serine the interaction between serine proteases and cytokines proteases expressed on the neutrophil surface may asso- amplifies the necessary signals that sustain neutrophil ciate with CD11b/CD18 (Mac-1), the major integrin on recruitment at sites of inflammation. the PMN surface (42). This concentrated expression of Using a well-defined acute experimental arthritis model serine proteases on the surface of cells could be impor- and the subcutaneous air pouch, we showed that the tant for the detachment and locomotion of PMNs, espe- absence of DPPI and active serine proteases totally abro- cially in an extracellular matrix–rich environment such as gated antibody-mediated acute arthritis, and significant- the synovium. It is likely that at specific inflammatory ly attenuated the response to zymosan and immune com- sites, excessive adhesion may actually interfere with effi- plex–induced inflammation in the subcutaneous air cient movement of neutrophils. Therefore, surface- pouch. In the arthritis model, immune complex forma- bound serine proteases may promote the release of PMNs tion led to the activation of the classical pathway of the from their integrin-mediated adhesion by cleaving the complement cascade and the generation of C5a. tethering association between integrins and their ligands. Although the activated complement component C5a is a In fact, purified human NE has been shown to cleave sur- potent chemoattractant, it has a short half-life. Other face ICAM-1 and recombinant soluble ICAM-1 (43), and cytokines and factors may be needed for maximal inflam- both NE and CG can cleave VCAM-1 (44). matory cell recruitment. NE has previously been shown In summary, this study has established that DPPI is to play a critical role in sustaining recruitment of PMNs the major enzyme activating neutrophil-derived serine in bullous pemphigoid induced in the mouse, another proteases in vivo. The importance of these proteases in model of antibody-mediated inflammation of the skin inflammatory processes was demonstrated by the dra- (25). In that report, the authors hypothesize that NE- matic protection against arthritis development in an in mediated extracellular matrix degradation products, such vivo model of acute inflammatory arthritis. These as elastin fragments, are chemotactic for neutrophils. observations also extend the role of neutrophil-derived Our present studies extend the role of PMN-derived ser- proteases in inflammation beyond degradation of

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